The role of intracellular alkalinization in the establishment of anterior neural fate in Xenopus.
Our previous work demonstrated that Xenopus ectoderm cells undergo an alkalinization in response to planar inductive signals during neural induction in explants. We have examined the role of intracellular alkalinization in the establishment of anterior neural fate. First, RT-PCR was used to examine neural-specific gene expression in planar explants in which the alkalinization is prevented by treatment with 4,4'-dihydrodiisothiocyanatostilbene-2,2'-disulfonate (H2DIDS). In explants cultured in the presence of H2DIDS, expression of NCAM and the anterior neural gene otx2 is greatly reduced or absent. Second, neural-specific gene expression was examined in isolates of uninduced animal cap ectoderm cultured in the presence of either methylamine or ammonium chloride. NCAM, otx2, and the anterior neural inducer noggin were expressed in alkalinized ectoderm, while the more posterior neural markers krox-20 and Hox B9 were undetectable. Expression of NCAM, otx2, and noggin was observed at stage 11 in both alkalinized ectoderm and the newly induced neural plate, suggesting that intracellular alkalinization could contribute to propagation of noggin signaling through the dorsal ectoderm. Alkalinization of uninduced ectoderm at stage 10.5 led to an upregulation of otx2 within 15 min. Activation of NCAM expression in alkalinized dissociated cells was identical to that observed in intact animal caps, indicating that alkalinization-mediated changes in gene expression do not require cell-cell contact. Finally, the effects of intracellular alkalinization on protein tyrosine phosphorylation were investigated using 2D gel electrophoresis and immunoblots probed with an antiphosphotyrosine antibody. Several phosphorylated protein detected in induced and alkalinized ectoderm were greatly reduced or absent in uninduced ectoderm, indicating that alkalinization elicits alterations in tyrosine phosphorylation similar to some of those observed during neural induction in vivo. Our results indicate that intracellular alkalinization plays a critical role in the activation of anterior neural-specific gene expression and that alkalinization may act by regulating the activity of a tyrosine kinase or phosphatase.
Uzman
J A
JA
Department of Biology, University of Houston, Texas 77204-5513, USA.
Patil
S
S
Uzgare
A R
AR
Sater
A K
AK
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins; D002450:Cell Communication; D046508:Culture Techniques; D004475:Ectoderm; D004627:Embryonic Induction / Q000235:genetics / Q000502:physiology*; D005775:Gastrula; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D018398:Homeodomain Proteins; D006863:Hydrogen-Ion Concentration; D008744:Methylamines / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000235:genetics; D009420:Nervous System / Q000166:cytology / Q000196:embryology*; D019006:Neural Cell Adhesion Molecules / Q000235:genetics; D051857:Otx Transcription Factors; D019000:Phosphotyrosine / Q000032:analysis; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D015534:Trans-Activators / Q000235:genetics; D014981:Xenopus; D029867:Xenopus Proteins; D000099176:Noggin Protein
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D000643:Ammonium Chloride; D002352:Carrier Proteins; D018398:Homeodomain Proteins; D008744:Methylamines; D009419:Nerve Tissue Proteins; D019006:Neural Cell Adhesion Molecules; D051857:Otx Transcription Factors; D019000:Phosphotyrosine; D011506:Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D029867:Xenopus Proteins; D000099176:Noggin Protein; C497661:Otx2 protein, Xenopus; C018564:dihydro-DIDS; C027451:methylamine